Species differences in the metabolism of trichloroethylene to the carcinogenic metabolites trichloroacetate and dichloroacetate

TOXICOLOGY

AND

APPLIED

PHARMACOLOGY

115,278-285

(1992)

Species Differences in the Metabolism of Trichloroethylene to the Carcinogenic Metabolites Trichloroacetate and Dichloroacetate’T* J. L. LARSON~ AND R. J. BULL College

of Pharmacy.

Il’ashingion

State

University.

Pullman,

Washing(on

99164

Received November 26. 199 I : accepted April 8, 1992

duced by carcinogenic doses of the chlorinated acetates makes SpeciesDifferences in the Metabolism of Trichloroethylene to the Carcinogenic Metabolites Trichloroacetate and Dichloroacetate. LARSON, J. L., AND BULL, R. J. (1992). Toxicol. Appl. Pharmacol. 115,278-285.

it highly likely that both compoundsplay a role in the induction . . of hepattc tumors in mice by TCE. Q 1992 Academic PM, IW.

Differing rates and extent of trichloroethylene (TCE) metabolism havebeenimplicated asbeingresponsiblefor varying sensitivities of mice and rats to the hepatocarcinogenic effects of TCE. Recent data indicate that the induction of hepatic tumors in mice may be attributed to the metabolites trichloroacetate (TCA) and/or dichloroacetate (DCA). The present study was directed at determining whether mice and rats varied in (1) the peakblood concentrations, (2) the area under the blood concentration over time curves (AUC) for TCE and metabolites in blood, and (3) the net excretion of TCE to thesemetabolitesin urine in the dose range used in the cancer bioassaysof TCE, and to contrast the kinetic parametersobservedfor TCE-derived TCA and DCA with those obtained following direct administration of TCA andDCA. Blood and urine sampleswerecollected over 72 hr from rats and mice after a single oral dose of TCE of 1.5 to 23 mmobkg. The AUC values from the blood concentration with time profiles of TCE, TCA, and trichloroethanol (TCOH) were similar for Sprague-Dawley rats and B6C3Fl mice. Likewise, the percentagesof initial TCE dose recovered as the urinary metabolites TCA and TCOH were comparable. Nevertheless,the peak blood concentrations of TCE, TCA, and TCOH observedin mice were much greater than those in rats, while the residencetime of TCE and metaboliteswasprolonged in rats relative to that of mice. DCA wasdetected in the blood of mice but not in rats. The blood concentrations of DCA observedin mice given a carcinogenic doseof TCE (15 mmol/kg) wereof the samemagnitudeasthoseobservedwith carcinogenic dosesof DCA. In conclusion, the net metabolism of TCE to TCA and TCOH was similar in rats and mice. The initial rates of metabolismof TCE to TCA, however, were much higher in mice,especiallyasthe TCE dosewasincreased,leadingto greater concentrations of TCA in blood. The fact that the blood concentrations of TCA and DCA in mice approximated those pro’ This work was supported by U.S. Air Force Grant AFOSR-86-0284 and by EPA Cooperative Agreement CR-8 152 16-OI. z Presented in part at the 29th Annual Meeting ofthe Society of Toxicology, February 1990, Miami. FL. 3 To whom reprint requests should be addressed at Chemical Industry Institute of Toxicology. P.O. Box I2 137, Research Triangle Park. NC 27709. 0041-008X/92 $5.00 Copyright All rights

6 1992 by Academic Press. Inc. of reproduction in any form reserved.

Trichloroethylene (TCE) has been and continues to be a commonly used industrial solvent. As such, it is a common ground and surface water contaminant (Murray and Riley, 1973; Coniglio et al., 1980). This occurrence has raised the possibility of health hazards to the consumers of these waters. TCE administered chronically increased the incidence of hepatocellular carcinoma in B6C3Fl mice when given by gavage in corn oil (NCI, 1976; NTP, 1990). The same studies found that TCE did not increase the incidence of tumors in livers of Osborne-Mendel or Fischer 344 rats, respectively. To understand the potential for development of liver cancer in humans exposed to TCE, the mechanism of tumor induction in mice must be elucidated. It has been postulated that trichloroacetate (TCA) is the metabolite of TCE which leads to liver tumor induction in mice (Elcombe, 1985; Fisher et al., 1991). Consistent with the observed species difference in hepatic tumorigenicity associated with TCE treatment, TCA is much more effective at inducing hepatic peroxisomes in mice than in rats (DeAngelo et a/., 1989). Although largely overlooked, dichloroacetate (DCA) is also a metabolite of TCE (Hathaway, 1980; Dekant et al.. 1984) and TCA (Larson and Bull, 1992) as well as a peroxisome proliferator in rodents (DeAngelo et al., 1989). Both TCA and DCA have been shown to increase the incidence of hepatic tumors in B6C3Fl mice when administered in drinking water (Herren-Freund ef af.. 1987; Bull ef al., 1990; DeAngelo et al., 199 1). In view of the hepatocarcinogenic effects elicited by both DCA and TCA, it is reasonable to conclude that one or both of these compounds are the proximate carcinogens in mice following administration of TCE. While there are studies on the urinary elimination of TCE and its metabolites and some attempt to identify blood levels (Prout et al., 1985; Green and Prout, 1985; Dekant et al., 1984, 1986), no study has been designed to examine the characteristics of both formation and elimination of metabolites at the high dose levels of TCE used in the NC1 (1976)

278

SPECIES DIFFERENCES

bioassay. These characteristics become especially important when trying to attribute the hepatocarcinogenic effect of TCE to a particular metabolite. To date, there have been several other metabolites of TCE identified in the urine of rodents. These include trichloroethanol (TCOH), the major urinary metabolite, found in both the free or glucuronidated form, oxalic acid, and N(hydroxyacetyl)aminoethanol (Dekant et al., 1984). While the anesthetic properties of TCOH are well known (Green, 1968), the possible carcinogenicity of the compound has not been addressed. In light of the fact that the only metabolites of TCE currently known to produce liver cancer are TCA and DCA, the current study evaluates more critically the role of the chlorinated acetates in the hepatocarcinogenic effects of TCE. This is accomplished by determining the peak blood concentrations (C,,,) and the areas under the blood concentration over time curves (AUC) of TCA and DCA achieved in the blood of rats and mice from administration of TCE and comparing these values to data developed in the accompanying paper, where carcinogenic doses of the chlorinated acetates were administered. METHODS Chemicals Analytical grade TCE was obtained from Fisher Scientific (Fair Lawn, NJ). TCA, TCOH, and DCA were obtained from Sigma Chemical Co. (St. Louis, MO). and used as standards to identify and quantitate metabolite levels. Polysorbate Tween 80 was also purchased from Sigma Chemical Co. Animals and Treatment Male Sprague-Dawley rats and male B6C3Fl mice, weighing 404 + 9 1 g and 26.4 i 3.2 g, respectively, were obtained as needed from Simonsen Laboratories (Gilroy. CA) and housed in an environmentally controlled animal room at 22-24°C with a relative humidity of 40-60% and a 12-hr light-dark cycle. Purina rodent chow and water were available ad libitum. Rats were fasted for 24 hr and mice for 4 hr prior to administration of the oral dose of TCE. In each experimental treatment group there were five to six animals receiving doses of TCE at 1.5, 4.5, and I5 mmol/kg for mice and 1.5, 4.5, and 23 mmol/kg for rats. The high dose for mice was lower than that given to rats due to the lethality of a 23 mmol/kg dose in mice. The doses of TCE were prepared fresh immediately prior to administration as suspensions in 1% aqueous Tween 80. The doses were given between 9:00 and 1 I:00 AM to minimize diurnal variations with the dose administered in a constant volume of 3 ml/kg to rats and 10 ml/kg to mice.

IN METABOLISM

279

OF TCE

following derivatization with diazomethane. The samples were analyzed using a Varian Model 3700 gas chromatograph (electron-capture detection) fitted with a 2-mm X 2-ft glass column packed with 0.1% AT 1000 on SO/l00 mesh Graphpac-GC (column temperature, 13O”C, nitrogen carrier gas flow 25 ml/min). Under these conditions the following retention times were recorded: TCE, 1.5 min; DCA, 2.8 min; TCOH, 4.3 min: and TCA, 5.7 min. Triplicate determinations of each sample were made. Urinalysis. The collection of urine was conducted with additional groups of six mice per dose level of TCE. Since the rats were not terminated until 72 hr post-TCE dosing, the rats used for the analysis of TCE and metabolites in blood were also used for the collection of urine. immediately following dosing with TCE, rats were housed individually in polycarbonate cages for collection of urine over the intervals O-24,24-48, and 48-72 hr. Mice were housed in specially constructed PVC cages designed to accommodate a single animal: urine was collected over the same intervals. Rodents were euthanized by decapitation at the end of the experiments. TCA, DCA, and TCOH were found to be stable in the urine for 24 hr; concentrations did not vary in urine spiked with these compounds and allowed to stand at room temperature for 24 hr. Urine was treated as described above in the analysis of blood for TCA. DCA. and free TCOH. Total urinary TCOH was determined following treatment of urine aliquots with /l-glucuronidase, as described in Green and Prout (1985). Standards were prepared by adding known amounts ofTCE. TCA. TCOH. and DCA to blood and urine of naive animals. The samples were analyzed as described above for blood and urinalysis. Under the conditions of the assay. the limits of detection were 15 nmol/ml for TCE, 15 nmol/ml for TCA, I nmol/ml for TCOH and 4 nmol/ml for DCA. Kinetic Anal.vsis The terminal elimination rate constants (KE) were calculated by plotting the log of the blood concentration over linear time and calculating the slope of the terminal, linear portion of the line. The elimination rate constant is obtained by KE = -2.3 slope. The value of the constant is expressed in hr-‘. The elimination half-life values (t,,*) for the chemicals in the blood were derived using the equation t,,z = 0.693/K,. The half-life is expressed in hours. The rate of formation of metabolites (KF) from the parent compound was calculated using the method of residuals to separate the two exponential forms (formation and elimination). Using the metabolite blood concentration over time curve, the rising portion of the blood concentration curve is subtracted from the extrapolated terminal elimination line to give a line with a slope providing an estimate of the KF. The unit values are hr-i. The volume of distribution ( Vd) of the parent compound was calculated from

TCE and Metabolite Anal.vsis Blood samples were collected from groups of five to six rats via the tail vein and from mice by decapitation at all time points. Thus, each rat provided serial blood concentrations, while single point determinations were made in mice, five to six mice per time point. Following administration of TCE, blood samples (200 to 300 ~1) were collected from rats at I, 2,4, 8, 12, 24,48, and 72 hr and from mice at 0.25.0.75, 2,4, 8, 24, 48, and 72 hr. Food was available to the animals after the sample was taken at Hour 2. TCE, TCA, DCA, and free TCOH (unconjugated) were determined by gas chromatography utilizing the ether extraction method of Prout et al. (I 985). TCA and DCA were detected as their methyl esters Blood

sampling.

Vd = Dose/CbO, where Cb” is the blood concentration at which the extrapolated terminal elimination line crosses the y-axis (blood concentration). The value of this parameter is expressed as milliliters per killogram. Clearance (CL) was calculated from the equation CL=Krx

I’d,

The value is expressed as milliliters per killogram per hour.

280

LARSON

AND

BULL RATS

MICE 500

400

g 3w ciz I 200 !

0

2

4

6

6

20

1W

0

4

2

6

Time

6

0

24 32 16 Time (hr)

6

(hr)

40

46

0 0

4

6

12

16

20

24

-0

6

16

Time (hr)

FIG. 1. Blood concentrations of TCE in mice (left) and rats (right) following single oral doses of TCE. Mice were given an oral dose of 1.5 (0). 4.5 (A). or I5 (0) mmol/kg TCE in Tween 80. Rats received either 1.5 (0). 4.5 (A). or 23 (0) mmol/kg TCE in Tween 80. Each point represents the mean value f SEM offive to six serially sampled rats or five to six individual mice. Insets illustrate the early time points.

RESULTS TCE Metabolism The blood concentration with time curves for TCE, TCA, and TCOH were significantly different in rats and mice (Figs. l-3). The peak blood concentrations of TCE and TCA were about 3 times greater and the free TCOH peak about 10

24

32

40

46

Time (hr)

FIG. 3. Blood concentrations of TCOH in mice (left) and rats (right) following single oral doses of TCE. Mice were given an oral dose of I .5 (0). 4.5 (A), or 15 (0) mmol/kg TCE in Tween 80. Rats received either 1.5 (0). 4.5 (A). or 23 (0) mmol/kg TCE in Tween 80. Each point represents the mean value + SEM of five to six serially sampled rats or five to six individual mice. Insets illustrate the early time points.

times greater in mice relative to that in rats. Blood concentrations for DCA in mice were only found in those given the 15 mmol/kg dose of TCE (Fig. 4). Blood concentrations of DCA in mice given the lower doses of TCE were not present in sufficient amounts to be quantifiably measurable. DCA

MICE RATS

MlCE

40

500

1 T

30

0

2

4

6

n

6

100 150 500 0

2

4

6

6

20

200

IO

0 0

12

24 Time

36

46 (hr)

60

72

0

12

24

36 Time

46

60

72

(hr)

FIG. 2. Blood concentrations of TCA in mice (left) and rats (right) following single oral doses of TCE. Mice were given an oral dose of 1.5 (0). 4.5 (A), or 15 (0) mmol/kg TCE in Tween 80. Rats received either 1.5 (0). 4.5 (A), or 23 (0) mmol/kg TCE in Tween 80. Each point represents the mean value * SEM of five to six serially sampled rats or five to six individual mice. Insets illustrate the early time points.

0

0

a

16

24

32

40

48

Time (hr) FIG. 4. Blood concentrations of DCA in mice following doses of 15 mmol/kg TCE in Tween 80. Each point represents value + SEM of five to six individual mice.

single oral the mean

SPECIES DIFFERENCES

was not found in the blood of treated rats at a detection limit of 4 nmol/ml, nor were any other ether-extractable chlorinated metabolites of TCE identified in blood samples. The blood concentration versus time curves also show the effect of dose on the peak concentrations of TCE, TCA, and TCOH. The peak concentrations of TCE in the blood were linearly related, in both species, to the dose of TCE administered (Fig. 1). In contrast, the increases in peak blood concentrations of the metabolites were considerably less than proportional to the increase in TCE dose in both species. The time to reach the peak blood concentrations of TCE and its metabolites was also markedly different between the two species. In mice, the peak blood concentration of TCE

0

10

5

15

20

25

0

5

10

15

20

25

0

5

10

15

20

25

TCE dose (mmolikg) FIG. 5. Blood AUC for TCE (top). TCA (middle), and TCOH (bottom) in rats (0) and mice (A) following administration of TCE. Values were calculated by applying the linear trapezoidal method to the blood concentration over time curves. Each point is the mean f SEM. The AUC was calculated for each individual rat and for each randomized mouse set.

IN METABOLISM

281

OF TCE

TABLE 1

Blood Kinetic Parameters for TCE Observed with a Single Oral Dose of TCE Dose (mmolfkg) 1.5 4.5 15 23

Species Mice Rats Mice Rats Mice Rats

1 (z-)

ii W/kg)

0.5

2.200 10,300

1.3 0.8 3.0

1.1 4.3

1.100 11.500 3,700 10,000

CL (ml/kg-hr) 3.260

5.150 1.037 2.700 7 173 _,--_ I.515

was reached within 15 min after TCE administration (Fig. 1). The peak TCE blood concentration in rats after a 1.5 mmol/kg TCE dose was reached within 1 hr, while increasing the doses resulted in progressive delays in the time to reach peak blood concentrations. Likewise, peak blood concentrations of TCA and TCOH were reached much earlier in mice than in rats (Fig. 2). Peak blood concentrations of TCA in the mice were reached within 45 min at the lowest dose of TCE, and at 8 hr at the two higher TCE doses. Peak blood concentrations of TCOH were reached rapidly in mice, within 2 hr at any given TCE dose (Fig. 3). In contrast, the time to reach peak blood concentrations of TCA in rats occurred at 8, 12, and 24 hr after dose of 1.5, 4.5. and 23 mmol/kg TCE, respectively (Fig. 2). A similar pattern was observed for TCOH in rats (Fig. 3). It is important to note that while the shape of the blood concentration over time curves for TCE were quite different between rats and mice, the AUC values for TCE were not (Fig. 5). On the other hand, the AUC values for TCA and TCOH were 1.5 to 2.5 times greater in mice than those in rats at all dose levels. The half-life of TCE was only mildly dose dependent in mice, but increased progressively in rats, suggesting saturated clearance of TCE in this species (Table 1). However, the apparent Vd was much greater in rats than that in mice. This led to comparable clearance rates in both species, despite the higher elimination rate for TCE in mice. The formation rates of TCA from TCE in mice were the same at doses of 4.5 and 15 mmol/kg TCE, although the half-life of TCA increased from 4.1 to 7.7 hr (Table 2). The I& value for TCA calculated in mice administered 1.5 mmol/ kg TCE was about twice that of the higher dose levels, although the half-life was about the same as that observed following the 4.5 mmol/kg dose of TCE. Similarly, in rats, the rate of TCA formation at a dose of 1.5 mmol/kg was twice that observed with the TCE doses of 4.5 and 23 mmol/ kg, and the half-life increased from 5.0 to 7.0 hr (Table 2). The decreased rate constant for formation of TCOH provides evidence of saturation of TCE metabolism in rats as the dose of TCE is increased above 1.5 mmol/kg (Table 2). An apparent plateau of blood concentrations of TCOH in

282

LARSON TABLE

TABLE

2

Blood Kinetic Parameters for Metabolities of TCE after a Single Oral Dose of TCE Dose (mmol/kg)

Species

AND BULL

1 (2,

KF (hr-‘)

3

Percentage of the Initial TCE Dose Recovered in the Urine as TCA or TCOH over a 0- to 72-Hr Period % of TCE dose as Dose (mmol/kg)

Species

TCOH

TCA

TCA 1.5 4.5 15 23

Mice Rats Mice Rats Mice Rats

5.3 5.0 4.1 7.0 7.1 7.0

0.42 0.24 0.27 0.13 0.27 0.13

Mice

4.6

4.5 15 23

Mice Rats Mice Rats Mice Rats

15 23

5.4 + 4.8 + 3.9 + 4.1 k 3.1 f 2.3 +

0.8 0.8 0.3 0.6 0.7 0.8

27.1 f 2.8 23.4 f 3.6 31.0 f 7.2 23.2 i 4.0 20.6 f 3.0 10.3 + 2.4

0.34

TCOH 1.5

4.5

Mice Rats Mice Rats Mice Rats

Note. Values represent means + standard errors.

DCA 15

1.5

0.5 2.0 0.7 4.1 2.7 5.3

NC” 0.78 1.3 0.22 0.75 NC

’ NC, not calculated.

rats administered the 23 mmol/kg dose of TCE prevented calculation of the KF value at this dose. The KF value was 0.2 hr-’ following a 4.5 mmol/kg dose of TCE, compared to 0.8 hr-’ at the 1.5 mmol/kg TCE dose. It is worth noting that the KF for TCOH in mice is sixfold greater than in that in rats at equivalent doses of 4.5 mmol/kg TCE. Despite the differences in the AUC values for TCA and TCOH in rats and mice, the extent of metabolite recovery in the urine was quite similar. The amount of metabolites recovered in urine as a percentage of initial TCE dose is presented in Table 3. At the 1.5 and 4.5 mmol/kg doses of TCE, the percentage of initial TCE dose that was recovered in the urine as either TCA or TCOH was much the same in rats as it was in mice. The percentage of initial dose of TCE recovered as TCA and TCOH decreased as the dose of TCE was increased in both species. Since it was necessary to dilute the urine samples to bring the concentrations of TCA and TCOH into a quantifiable range, it was not possible to detect other minor chlorinated ether-extractable metabolites of TCE using this methodology. TCA and DCA Metabolism The peak blood concentrations and AUC values for TCA in rats and mice administered TCE were compared to the same parameters collected following direct administration of TCA (Larson and Bull, 1992) (Table 4). In rats, the peak blood concentration of TCA following administration of 23

mmol/kg TCE corresponded to an oral dose of approximately 0.20 mmol/kg TCA (determined by interpolation between doses of 0.12 and 0.60 mmol/kg TCA). On the other hand, the AUC value in rats given 23 mmol/kg TCE approximated that produced by an oral dose of 0.60 mmol/kg TCA. In mice given 15 mmol/kg TCE, the peak blood concentration of TCA was roughly the same as that observed following an oral dose of 0.60 mmol/kg TCA, while the AUC value for TCA following this high dose of TCE was twice as great as that which was observed in mice administered 0.60 mmol/ kg TCA. The peak blood concentrations of DCA resulting from an oral dose of 15 or 23 mmol/kg TCE to mice and rats, respectively, are presented in Table 5 along with the peak blood concentrations of DCA observed in rodents administered oral doses of either DCA or TCA. The peak blood concentration for DCA in mice given the highest dose of TCE corresponded to an oral dose of DCA in excess of 0.78 mmol/

TABLE

4

Peak Blood Levels and Blood AUC Values for TCA Following Oral Administration of a Single Dose of TCE or TCA” Peak blood levelb in

AUC? in

Treatment (mmol/kg)

Mice

Rats

Mice

Rats

1.5 4.5 15 23 0.12 0.60

216 624 753 ND 233 783

81 151 NDd 314 226 1233

2.5 9.3 14.3 ND 2.0 7.2

1.5 4.1 ND 10.3 2.5 10.0

TCE TCE TCE TCE TCA TCA

a Values for peak blood levels and AUC following an oral dose of TCA were obtained from the accompanying paper (Larson and Bull, 1992). b Blood concentrations are reported in nmol/ml. ’ AUC values are reported in rmol-hr/ml. d ND indicates that this dose of TCE was not used in this species.

SPECIES DIFFERENCES

TABLE 5 Peak Blood Levels of DCA following Administration of TCE, TCA, or DCA” Peak blood levels’ in Treatment (mmoljkg) 15 ‘3 0.12 0.60 0.16 0.78

TCE TCE TCA TCA DCA DCA

Mice

Rats

35 ND 2 5 4 20

ND’ <@ 2 31 15 378

’ Values for peak blood levels following an oral dose of TCA and DCA were obtained from the accompanying paper (Larson and Bull, 1992). b Blood concentrations are reported in nmol/ml. ’ ND indicates that this dose of TCE was not used in this species. ‘The detection limit for DCA in the blood was 4 nmol/ml.

kg DCA, while the lack of quantifiable levels of DCA in rats suggests much lower exposures of rats to this metabolite. DISCUSSION The data from this study provide new insights into the species-specific metabolism of high doses of TCE. On the one hand, if the metabolism of TCE is evaluated solely on the basis of collection and quantitation of urinary metabolites, then the net amount of TCE apparently metabolized would appear to be similar in rats and mice. Indeed, quantitation of urinary metabolites of TCE as an estimation of dose has been reported in previous studies and suggested as a noninvasive estimation of TCE dose (Axelson et al., 1978; Monster, 1984). However, our data show that the amount of urinary metabolites does not necessarily reflect the internal exposure to metabolites. Likewise, evaluation of the extent of TCE exposure employing a single time point determination of blood concentrations would also provide a distorted picture of actual TCE and metabolite exposure, depending on the time point sampled. Mice metabolized TCE at a much faster rate relative to that of rats, thereby producing significantly higher blood concentrations of TCA and TCOH at comparable doses of TCE at earlier time points. The percentage of initial dose of TCE recovered in urine as TCA was not significantly different in mice and rats given 1.5 or 4.5 mmol/kg TCE. Similar recoveries of TCA (2 to 7%) in the urine and a lack of species difference in the net conversion of TCE to TCA have been noted by other investigators (Green and Prout, 1985: Dekant et al., 1986; Rouisse and Chakrabarti, 1986). The amount of TCOH recovered in the urine as a percentage of initial dose was also similar in rats and mice administered 1.5 or 4.5 mmol/kg TCE. Increasing the dose of TCE in rats to 23 mmol/kg did result in a 50% decrease in the percentage of the initial TCE dose

IN METABOLISM

OF TCE

283

recovered as TCOH. This evidence that metabolism of TCE to TCOH was saturated at these dose levels is consistent with the results of Dekant et al. (1984, 1986). There is a significant difference in absorption kinetics for TCE in mice and rats, evidenced by the delay in time to peak blood levels and prolonged residence times for TCE in rats. The slower absorption of TCE from the GI tract of rats might be responsible for the observed large Vi, and subsequent high CL values in rats. A delay in absorption of TCE would increase Vn due to the lower initial blood concentrations of TCE. Indeed, D’Souza et al. (1985) calculated a I/;, of 50-70 ml/kg when rats were given TCE by iv injection, which is substantially lower than our calculated V,,. The longer period of time that TCE remained in the blood of rats resulted in similar AUC’s for TCA and TCOH in rats and mice at the low 1.5 mmol/kg dose. Increasing the TCE dose to 4.5 mmol/kg, however, markedly increased the blood AUC values for both TCA and TCOH to levels threefold higher in mice than that in to rats. Consequently. the difference between rats and mice in metabolism of TCE becomes more marked as the dose of TCE approaches those doses, 4 and 8 mmol/kg in rats and 9 and 18 mmol/kg in mice, used in the carcinogenesis bioassays of TCE (NCI, 1976; NTP, 1990). The data in the current study implies that it is not the total net conversion of TCE to metabolites that is important in the species difference to induction of hepatic tumors as previously suggested (Elcombe, 1985) but rather the greater peak blood concentrations of metaboIites attained in mice. On the basis of the threshold response to induction of peroxisomes observed in rodents administered TCA, Elcombe rf al. (1985) postulated that the metabolism of TCE will generate an amount of TCA sufficient to induce peroxisomes only in mice (Table 6). Their threshold dose of TCA required for peroxisome induction was 0.3 mmol/kg. Our data strongly support the hypothesis that the peak blood concentrations of TCA are the determining factor in the induction of peroxisomes and possibly, hepatocarcinogenesis. To illustrate, the AUC data in our study for TCA arising from metabolism of the highest TCE dose in rats corresponds to a dose of TCA great enough to induce peroxisomes, that is, a dose of 0.6 mmol/kg. In contrast, the peak blood concentration of TCA in rats given the 23 mmol/kg dose of TCE corresponded to an oral dose of less than 0.2 mmol/kg TCA. Assuming the same slight upward trend in TCA peak blood concentrations with increasing TCE doses, the peak blood concentrations of TCA necessary to induce peroxisome proliferation could not be reached with nonlethal doses of TCE. This difference in the AUC and peak concentration values as estimates of internal dose of TCA is most likely due to the prolonged residence time for TCE in rats. Thus, while metabolism of TCE in rats is saturated and blood concentrations plateau, the AUC values continue to increase due

284

LARSON

TABLE 6 Comparison of the Average Daily Dose of TCE, TCA, and DCA Required to Increase the Incidence of Liver Peroxisomes and Tumors Minimum daily dose’] (mmol/kg) in Mice

Rats

Daily dose of TCE needed to induce Peroxisome Liver Tumors

0.46

None’ None

Daily dose of TCA needed to induce Peroxisome Liver tumors

0.8

Daily dose of DCA needed to induce Peroxisome Liver tumors

lhd

4.2”

1.08

None

2.0’ 1.2*

5.2e 2.3h

’ Lowest daily dose found in the literature. b Elcombe ( 1985). ’ No induction of peroxisomes or tumors have been reported in rats. d NTP (1990). ’ DeAngelo et al. (1989). ‘Daily doses of 0.3 mmol/kg TCA given in corn oil induce peroxisomes (Elcombe. 1985). while TCA in the drinking water at average daily doses of 4.2 mmol/kg does not (DeAngelo et al., 1989). g Bull et al. (1990). h An increase in enzyme altered foci observed at this dose level (DeAngelo and Daniel, 1992).

to the prolonged absorptive phase for TCE to body tissues and subsequent release and metabolism. While peak blood concentrations of TCA remained relatively low in rats, the peak blood concentrations of TCA in mice exceeded those expected from the threshold dose of 0.3 mmol/kg TCA at a TCE dose as low as 4.5 mmol/kg (Table 4). It should be pointed out that DeAngelo et al. ( 1989) also found that equivalent daily doses of TCA in drinking water were more effective in inducing peroxisomes (both numbers and palmitoyl CoA oxidation) in mice than in rats (Table 6). Thus, the data in this study and the accompanying paper provide evidence for the hypothesis that TCA-induced peroxisome proliferation is reliant on peak blood concentrations. The blood concentrations of DCA resulting from the high dose of TCE in mice may also be a factor in the carcinogenic response. The blood levels of DCA observed in mice given the high dose of 15 mmol/kg TCE corresponded to those blood concentrations observed following an oral bolus dose of 0.78 mmol/kg DCA (Table 5). An average daily dose of 1.2 mmol/kg has been previously shown to induce hepatic tumors in B6C3Fl mice (Table 6). These blood concentrations of DCA might be the result of an inhibition of elimination of DCA at this high TCE dose. This is supported by two observations. First, comparisons of the t iI2 value for TCE-

AND BULL

derived DCA with the f,,? for orally administered DCA clearly shows a difference in half-lives. The residence time for DCA in the body is prolonged when derived from TCE metabolism. In contrast, the elimination of DCA, when administered by oral gavage, is extremely rapid, rivaling that of TCE itself (Larson and Bull, 1992). Second, quantifiable blood concentrations of DCA were observed only with the 15 mmol/kg dose of TCE in mice. Since this dose reflects only a threefold increase in TCE dose, a linear relationship of metabolism of TCE to DCA should have resulted in measurable amounts of DCA at the 4.5 mmol/kg dose of TCE, but did not. DCA was not detected in the blood of rats even at doses of TCE up to 23 mmol/kg. The detection limit of the assay for DCA in blood was 4 nmol/ml in rodents administered TCE. If one assumes that this was the peak blood concentration of DCA in TCE-treated rats, then the corresponding oral dose of DCA was substantially less than 0.16 mmol/kg (Table 5); this appears to be a noncarcinogenic dose of DCA (Table 6). Given the demonstration that measurable quantities of DCA were formed only in mice administered the high dose of TCE, it remains possible that such a metabolite could contribute to the hepatocarcinogenicity of TCE. Evaluating this possibility requires more sensitive measures of the rate of DCA formation at lower doses of TCE. It is important to point out that the kinetic data for TCA and DCA reported in the accompanying paper was collected in F344 rats and used in this paper to estimate the amounts of chlorinated acetates formed from the metabolism of TCE in Sprague-Dawley rats. The switch in strain from SpragueDawley to F344 rats was done because carcinogenesis data on DCA and TCA are currently being investigated in the F344 rat (DeAngelo and Daniel, 1992). Preliminary results obtained from an ongoing investigation of TCE metabolism in the F344 rat in our laboratory indicate that there are no substantial differences in the kinetics of TCA formation or elimination following TCE treatment in these two strains. As with the Sprague-Dawley rat, DCA formation from the metabolism of TCE was not measurable in the blood of F344 rats. Therefore, we believe the estimates of TCA and DCA resulting from the metabolism of TCE from different strains is justified. In conclusion, this study demonstrates that mice administered high doses of TCE are exposed to internal doses of TCA that are comparable to those achieved with doses of TCA previously demonstrated to result in hepatic tumors (Bull et al., 1990). In contrast, the peak blood concentrations of TCA arising from a 23 mmol/kg dose of TCE are substantially below those required to induce peroxisome proliferation with administration of TCA. The fact that blood concentrations of DCA in mice given the high dose of TCE were also quite comparable to those blood concentrations of DCA produced by carcinogenic doses of that compound

SPECIES DIFFERENCES

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